Pathological tissue detection and treatment employing targeted benzoindole optical agents

ABSTRACT

Novel tumor specific phototherapeutic and photodiagnostic agents are disclosed. The compounds consist of a carbocyanine dye for visualization, photosensitizer for photodynamic treatment, and tumor receptor-avid peptide for site-specific delivery of the probe and phototoxic agent to diseased tissues. A combination of these elements takes full advantage of the unique and efficient properties of each component for an effective patient care management.

This application is a Divisional of U.S. patent application Ser. No.09/978,725 filed Oct. 17, 2001, now U.S. Pat. No. 6,761,878 andexpressly incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

This invention relates to novel dye-bioconjugates for use in diagnosisand therapy, particularly novel compositions of cyanine dyebioconjugates of bioactive molecules.

BACKGROUND OF THE INVENTION

Cancer will continue to be a primary cause of death for the foreseeablefuture, but early detection of tumors would improve patient prognosis(R. T. Greenlee et al., Cancer statistics, 2000, CA Cancer J. Clin.,2000, 50, pp. 7–33). Despite significant advances in current methods forthe diagnosis of cancer, physicians still rely on the presence of apalpable tumor mass. At this, however, the many benefits of earlymedical intervention may have been already compromised.

Photodiagnosis and/or phototherapy has a great potential to improvemanagement of cancer patient (D. A. Benaron and D. K. Stevenson, Opticaltime-of-flight and absorbance imaging of biologic media, Science, 1993,259, pp. 1463–1466; R. F. Potter (Series Editor), Medical opticaltomography: functional imaging and monitoring, SPIE Optical EngineeringPress, Bellingham, 1993; G. J. Tearney et al., In vivo endoscopicoptical biopsy with optical coherence tomography, Science, 1997, 276,pp. 2037–2039; B. J. Tromberg et al., Non-invasive measurements ofbreast tissue optical properties using frequency-domain photonmigration, Phil. Trans. Royal Society London B, 1997, 352, pp. 661–668;S. Fantini et al., Assessment of the size, position, and opticalproperties of breast tumors in vivo by non-invasive optical methods,Appl. Opt., 1998, 37, pp. 1982–1989; A. Pelegrin et al.,Photoimmunodiagnosis with antibody-fluorescein conjugates: in vitro andin vivo preclinical studies, J. Cell Pharmacol., 1992, 3, pp. 141–145).These procedures use visible or near infrared light to induce thedesired effect. Both optical detection and phototherapy have beendemonstrated to be safe and effective in clinical settings andbiomedical research (B. C. Wilson, Optical properties of tissues,Encyclopedia of Human Biology, 1991, 5, 587–597; Y-L. He et al.,Measurement of blood volume using indocyanine green measured withpulse-spectrometry: Its reproducibility and reliability, Critical CareMedicine, 1998, 26, pp. 1446–1451; J. Caesar et al., The use ofIndocyanine green in the measurement of hepatic blood flow and as a testof hepatic function, Clin. Sci., 1961, 21, pp. 43–57; R. B. Mujumdar etal., Cyanine dye labeling reagents: Sulfoindocyanine succinimidylesters, Bioconjugate Chemistry, 1993, 4, pp. 105–111; U.S. Pat. No.5,453,505; Eric Hohenschuh, et al., Light imaging contrast agents, WO98/48846; Jonathan Turner, et al., Optical diagnostic agents for thediagnosis of neurodegenerative diseases by means of near infra-redradiation, WO 98/22146; Kai Licha, et al., In-vivo diagnostic process bynear infrared radiation, WO 96/17628; Robert A. Snow, et al., Compounds,WO 98/48838].

Dyes are important to enhance signal detection and/or photosensitizingof tissues in optical imaging and phototherapy. Previous studies haveshown that certain dyes can localize in tumors and serve as a powerfulprobe for the detection and treatment of small cancers (D. A. Bellnieret al., Murine pharmacokinetics and antitumor efficacy of thephotodynamic sensitizer 2-[1-hexyloxyethyl]-2-devinylpyropheophorbide-a, J. Photochem. Photobiol., 1993, 20, pp. 55–61; G. A.Wagnieres et al., In vivo fluorescence spectroscopy and imaging foroncological applications, Photochem. Photobiol., 1998, 68, pp. 603–632;J. S. Reynolds et al., Imaging of spontaneous canine mammary tumorsusing fluorescent contrast agents, Photochem. Photobiol., 1999, 70, pp.87–94). However, these dyes do not localize preferentially in malignanttissues.

Efforts have been made to improve the specificity of dyes to malignanttissues by conjugating dyes to large biomolecules (A. Pelegrin, et al.,Photoimmunodiagnosis with antibody-fluorescein conjugates: in vitro andin vivo preclinical studies, J. Cell Pharmacol., 1992, 3, pp. 141–145;B. Ballou et al., Tumor labeling in vivo using cyanine-conjugatedmonoclonal antibodies, Cancer Immunol. Immunother., 1995, 41, pp.257–263; R. Weissleder et al., In vivo imaging of tumors withprotease-activated near-infrared fluorescent probes, Nature Biotech.,1999, 17, pp. 375–378; K. Licha et al., New contrast agents for opticalimaging: Acid-cleavable conjugates of cyanine dyes with biomolecules,Proc. SPIE, 1999, 3600, pp. 29–35). Developing a dye that can combinethe roles of tumor-seeking, diagnostic, and therapeutic functions hasbeen very difficult for several reasons. The dyes currently in uselocalize in tumors by a non-specific mechanism that usually relies onthe lipophilicity of the dye to penetrate the lipid membrane of thecell. These lipophilic dyes require several hours or days to clear fromnormal tissues, and low tumor-to-normal tissue ratios are usuallyencountered. Furthermore, combining photodynamic properties withfluorescence emission needed for the imaging of deep tissues requires amolecule that must compromise either the photosensitive effect of thedye or the fluorescence quantum yield. Photosensitivity of phototherapyagents relies on the transfer of energy from the excited state of theagent to surrounding molecules or tissues, while fluorescence emissiondemands that the excitation energy be emitted in the form of light (T.J. Dougherty et al., Photoradiation therapy II: Cure of animal tumorswith hematoporphyrin and light, Journal of National Cancer Institute,1978, 55, pp. 115–121). Therefore, compounds and compositions that haveoptimal tumor-targeting ability to provide a highly efficientphotosensitive agent for treatment of tumors are needed. Such agentswould exhibit enhanced specificity for tumors and would also haveexcellent photophysical properties for optical detection.

Each of the references previously disclosed is expressly incorporated byreference herein in its entirety.

SUMMARY OF THE INVENTION

The invention is directed to a composition for a carbocyanine dyebioconjugate. The bioconjugate consists of three components: 1) a tumorspecific agent, 2) a photosensitizer (phototherapy) agent, and 3) aphotodiagnostic agent. The inventive bioconjugates use the multipleattachment points of carbocyanine dye structures to incorporate one ormore receptor targeting and/or photosensitive groups in the samemolecule. The composition may be used in various biomedicalapplications.

The invention is also directed to a method for performing a diagnosticand therapeutic procedure by administering an effective amount of thecomposition of the cyanine dye bioconjugate to an individual. The methodmay be used in various biomedical applications, such as imaging tumors,targeting tumors with anti-cancer drugs, and performing laser guidedsurgery.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. shows representative structures of the inventive compounds.

FIG. 2 shows images taken at two minutes and 30 minutes post injectionof indocyanine green into rats with various tumors.

FIG. 3 shows fluorescent images of a CA20948 tumor bearing rat taken atone and 45 minutes post administration of cytate.

FIG. 4 is a fluorescent image of a CA20948 tumor bearing rat taken at 27hours post administration of cytate.

FIG. 5 shows fluorescent images of ex-vivo tissues and organs from aCA20948 tumor bearing rat at 27 hours post administration of cytate.

FIG. 6 is a fluorescent image of an AR42-J tumor bearing rat taken at 22hours post administration of bombesinate.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to novel compositions comprising cyanine dyeshaving a general formula 1

wherein W₁ and W₂ may be the same or different and are selected from thegroup consisting of —CR¹⁰R¹¹, —O—, —NR¹², —S—, and —Se; Y₁, Y₂, Z₁, andZ₂ are independently selected from the group consisting of hydrogen,tumor-specific agents, phototherapy agents, —CONH-Bm, —NHCO-Bm,—(CH₂)_(a)—CONH-Bm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH-Bm, —(CH₂)a—NHCO-Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO-Bm, —(CH₂)_(a)—N(R¹²)—(CH₂)_(b)—CONH-Bm,—(CH₂)_(a)—N(R¹²)—(CH₂)_(c)—NHCO-Bm,—(CH₂)_(a)—N(R¹²)—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH-Bm,—(CH₂)_(a)—N(R¹²)—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO-Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R¹²)—(CH₂)_(a)—CONH-Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R¹²)—(CH₂)_(a)—NHCO-Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R¹²)—CH₂—(CH₂OCH₂)_(d)—CONH-Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R¹²)—CH₂—(CH₂OCH₂)_(d)—NHCO-Bm, —CONH-Dm,—NHCO-Dm, —(CH₂)_(a)—CONH-Dm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH-Dm,—(CH₂)_(a)—NHCO-Dm, —CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO-Dm,—(CH₂)_(a)—N(R¹²)—(CH₂)_(b)—CONH-Dm,—(CH₂)_(a)—N(R¹²)—(CH₂)_(c)—NHCO-Dm,—(CH₂)_(a)—N(R¹²)—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH-Dm,—(CH₂)_(a)—N(R¹²)—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO-Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R¹²)—(CH₂)_(a)—CONH-Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R¹²)—(CH₂)_(a)—NHCO-Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R¹²)—CH₂—(CH₂OCH₂)_(d)—CONH-Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R¹²)—CH₂—(CH₂OCH₂)_(d)—NHCO-Dm, —(CH₂)_(a)—NR¹²R¹³, and —CH₂(CH₂OCH₂)_(b)—CH₂N R¹²R¹³; K₁ and K₂ are independentlyselected from the group consisting of C₁–C₃₀ alkyl, C₅–C₃₀ aryl, C₁–C₃₀alkoxyl, C₁–C₃₀ polyalkoxyalkyl, C₁–C₃₀ polyhydroxyalkyl, C₅–C₃₀polyhydroxyaryl, C₁–C₃₀ aminoalkyl, saccharides, peptides,—CH₂(CH₂OCH₂)_(b)—CH₂—, —(CH₂)_(a)—CO—, —(CH₂)_(a)—CONH—,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—, —(CH₂)_(a)—NHCO—,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—, —(CH₂)_(a)—O—, and —CH₂—(CH₂OCH₂)_(b)—CO—;X₁ and X₂ are single bonds, or are independently selected from the groupconsisting of nitrogen, saccharides, —CR¹⁴—, —CR¹⁴R¹⁵, —NR¹⁶R¹⁷; C₅–C₃₀aryl; Q is a single bond or is selected from the group consisting of—O—, —S—, —Se—, and —NR¹⁸; a₁ and b₁ independently vary from 0 to 5; R¹to R¹³, and R¹⁸ are independently selected from the group consisting ofhydrogen, C₁–C₁₀ alkyl, C₅–C₂₀ aryl, C₁–C₁₀ alkoxyl, C₁–C₁₀polyalkoxyalkyl, C₁–C₂₀ polyhydroxyalkyl, C₅–C₂₀ polyhydroxyaryl, C₁–C₁₀aminoalkyl, cyano, nitro, halogens, saccharides, peptides,—CH₂(CH₂OCH₂)_(b)—CH₂—OH, —(CH₂)_(a)—CO₂H, —(CH₂)_(a)—CONH-Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH-Bm, —(CH₂)_(a)—NHCO-Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO-Bm, —(CH₂)_(a)—OH and—CH₂—(CH₂OCH₂)_(b)—CO₂H; R¹⁴ to R¹⁷ are independently selected from thegroup consisting of hydrogen, C₁–C₁₀ alkyl, C₅–C₂₀ aryl, C₁–C₁₀ alkoxyl,C₁–C₁₀ polyalkoxyalkyl, C₁–C₂₀ polyhydroxyalkyl, C₅–C₂₀ polyhydroxyaryl,C₁ –C₁₀ aminoalkyl, saccharides, peptides, —CH₂(CH₂OCH₂)_(b)—CH₂—,—(CH₂)_(a)—CO—, —(CH₂)_(a)—CONH—, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—,—(CH₂)_(a)—NHCO—, —CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—, —(CH₂)_(a)—O—, and—CH₂—(CH₂OCH₂)_(b)—CO—; Bm and Dm are independently selected from thegroup consisting of bioactive peptides, proteins, cells, antibodies,antibody fragments, saccharides, glycopeptides, peptidomimetics, drugs,drug mimics, hormones, metal chelating agents, radioactive ornonradioactive metal complexes, echogenic agents, photoactive molecules,and phototherapy agents (photosensitizers); a and c independently varyfrom 1 to 20; b and d independently vary from 1 to 100.

The invention also relates to the novel composition comprisingcarbocyanine dyes having a general formula 2

wherein W₁, W₂, Y₁, Y₂, Z₁, Z₂, K₁, K₂, Q, X₁, X₂, a₁, and b₁ aredefined in the same manner as in Formula 1; and R¹⁹ to R³¹ are definedin the same manner as R¹ to R⁹ in Formula 1.

The invention also relates to the novel composition comprisingcarbocyanine dyes having a general formula 3

wherein A₁ is a single or a double bond; B₁, C₁, and D₁ areindependently selected from the group consisting of —O—, —S—, —Se—, —P—,—CR¹⁰R¹¹, —CR¹¹, alkyl, NR¹², and —C═O; A₁, B₁, C₁, and D₁ may togetherform a 6- to 12-membered carbocyclic ring or a 6- to 12-memberedheterocyclic ring optionally containing one or more oxygen, nitrogen, orsulfur atoms; and W₁, W₂, Y₁, Y₂, Z₁, Z₂, K₁, K₂, X₁, X₂, a₁, b₁, and R¹to R¹² are defined in the same manner as in Formula 1.

The present invention also relates to the novel composition comprisingcarbocyanine dyes having a general formula 4

wherein A₁, B₁, C₁, and D₁ are defined in the same manner as in Formula3; W₁, W₂, Y₁, Y₂, Z₁, Z₂, K₁, K₂, X₁, X₂, a₁, and b₁ are defined in thesame manner as in Formula 1; and R¹⁹ to R³¹ are defined in the samemanner as R¹ to R⁹ in Formula 1.

The inventive bioconjugates use the multiple attachment points ofcarbocyanine dye structures to incorporate one or more receptortargeting and/or photosensitive groups in the same molecule. Morespecifically, the inventive compositions consist of three componentsselected for their specific properties. One component, a tumor specificagent, is for targeting tumors. A second component, which may be aphotosensitizer, is a phototherapy agent. A third component is aphotodiagnostic agent.

Examples of the tumor targeting agents are bioactive peptides such asoctreotate and bombesin (7–14) which target overexpressed receptors inneuroendocrine tumors. An example of a phototherapy agent is2-[1-hexyloxyethyl]-2-devinylpyro-pheophorbide-a (HPPH, FIG. 1D, T=OH).Examples of photodiagnostic agents are carbocyanine dyes which have highinfrared molar absorbtivities (FIG. 1A-C). The invention provides eachof these components, with their associated benefits, in one molecule foran optimum effect.

Such small dye biomolecule conjugates have several advantages overeither nonspecific dyes or the conjugation of probes or photosensitivemolecules to large biomolecules. These conjugates have enhancedlocalization and rapid visualization of tumors which is beneficial forboth diagnosis and therapy. The agents are rapidly cleared from bloodand non-target tissues so there is less concern for accumulation and fortoxicity. A variety of high purity compounds may be easily synthesizedfor combinatorial screening of new targets, e.g., to identify receptorsor targeting agents, and for the ability to affect the pharmacokineticsof the conjugates by minor structural changes.

The inventive compositions are useful for various biomedicalapplications. Examples of these applications include, but are notlimited to: detecting, imaging, and treating of tumors; tomographicimaging of organs; monitoring of organ functions; performing coronaryangiography, fluorescence endoscopy, laser guided surgery; andperforming photoacoustic and sonofluorescent methods.

Specific embodiments to accomplish some of the aforementioned biomedicalapplications are given below. The inventive dyes are prepared accordingthe methods well known in the art.

In two embodiments, the inventive bioconjugates have the formulas 1 or 2where W₁ and W₂ may be the same or different and are selected from thegroup consisting of —C(CH₃)₂, —C((CH₂)_(a)OH)CH₃, —C((CH₂)_(a)OH)₂,—C((CH₂)_(a)CO₂H)CH₃, —C((CH₂)_(a)CO₂H)₂, —C((CH₂)_(a)NH₂)CH₃,—C((CH₂)_(a)NH₂)₂, —C((CH₂)_(a)NR¹²R¹³)₂, —NR¹², and —S—; Y₁ and Y₂ areselected from the group consisting of hydrogen, tumor-specific agents,—CONH-Bm, —NHCO-Bm, —(CH₂)_(a)—CONH-Bm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH-Bm,—(CH₂)_(a)—NHCO-Bm, —CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO-Bm, —(CH₂)_(a)—NR¹²R¹³,and —CH₂(CH₂OCH₂)_(b)—CH₂NR¹²R¹³; Z₁ and Z₂ are independently selectedfrom the group consisting of hydrogen, phototherapy agents, —CONH-Dm,—NHCO-Dm, —(CH₂)_(a)—CONH-Dm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH-Dm,—(CH₂)_(a)—NHCO-Dm, —CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO-Dm, —(CH₂)_(a)—N R¹²R¹³,and —CH₂(CH₂OCH₂)_(b)—CH₂N R¹²R³; K₁ and K₂ are independently selectedfrom the group consisting of C₁–C₁₀ alkyl, C₅–C₂₀ aryl, C₁–C₂₀ alkoxyl,C₁–C₂₀ aminoalkyl, —(CH₂)_(a)—CO—, —(CH₂)_(a)—CONH,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—, —(CH₂)_(a)—NHCO—,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—, and —CH₂—(CH₂OCH₂)_(b)—CO—; X₁ and X₂ aresingle bonds, or are independently selected from the group consisting ofnitrogen, —CR¹⁴—, —CR¹⁴R¹⁵, and —NR¹⁶R¹⁷; Q is a single bond or isselected from the group consisting of —O—, —S—, and —NR¹⁸; a₁ and b₁independently vary from 0 to 3; Bm is selected from the group consistingof bioactive peptides containing 2 to 30 amino acid units, proteins,antibody fragments, mono- and oligosaccharides; Dm is selected from thegroup consisting of photosensitizers, photoactive molecules, andphototherapy agents; a and c independently vary from 1 to 20; and b andd independently vary from 1 to 100.

In two other embodiments, the bioconjugates according to the presentinvention have the formulas 3 or 4 wherein W₁ and W₂ may be the same ordifferent and are selected from the group consisting of —C(CH₃)₂,—C((CH₂)_(a)OH)CH₃, —C((CH₂)_(a)OH)₂, —C((CH₂)_(a)CO₂H)CH₃,—C((CH₂)_(a)CO₂H)₂, —C((CH₂)_(a)NH₂)CH₃, —C((CH₂)_(a)NH₂)₂,—C((CH₂)_(a)NR¹²R³)₂, —NR², and —S—; Y₁ and Y₂ are selected from thegroup consisting of hydrogen, tumor-specific agents, —CONH-Bm, —NHCO-Bm,—(CH₂)_(a)—CONH-Bm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH-Bm, —(CH₂)_(a)—NHCO-Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO-Bm, —(CH₂)_(a)—NR¹²R¹³, and—CH₂(CH₂OCH₂)_(b)—CH₂NR¹²R¹³; Z₁ and Z₂ are independently selected fromthe group consisting of hydrogen, phototherapy agents, —CONH-Dm,—NHCO-Dm, —(CH₂)_(a)—CONH-Dm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH-Dm,—(CH₂)_(a)—NHCO-Dm, —CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO-Dm, —(CH₂)_(a)—N R¹²R¹³,and —CH₂(CH₂OCH₂)_(b)—CH₂N R¹²R¹³; K₁ and K₂ are independently selectedfrom the group consisting of C₁–C₁₀ alkyl, C₅–C₂₀ aryl, C₁–C₂₀ alkoxyl,C₁–C₂₀ aminoalkyl, —(CH₂)_(a)—CO—, —(CH₂)_(a)—CONH,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—, —(CH₂)_(a)—NHCO—,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—, and —CH₂—(CH₂OCH₂)_(b)—CO—; X₁ and X₂ aresingle bonds or are independently selected from the group consisting ofnitrogen, —CR¹⁴—, —CR¹⁴R¹⁵, and —NR¹⁶R¹⁷; A₁ is a single or a doublebond; B₁, C₁, and D₁ are independently selected from the groupconsisting of —O—, —S, —CR¹¹, alkyl, NR¹², and —C═O; A₁, B₁, C₁, and D₁may together form a 6- to 12-membered carbocyclic ring or a 6- to12-membered heterocyclic ring optionally containing one or more oxygen,nitrogen, or sulfur atoms; a₁ and b₁ independently vary from 0 to 3; Bmis selected from the group consisting of bioactive peptides containing 2to 30 amino acid units, proteins, antibody fragments, mono- andoligosaccharides; bioactive peptides, protein, and oligosaccharide; Dmis selected from the group consisting of photosensitizers, photoactivemolecules, and phototherapy agents; a and c independently vary from 1 to20; and b and d independently vary from 1 to 100.

In one embodiment of the invention, the dye-biomolecule conjugates areuseful for optical tomographic, endoscopic, photoacoustic andsonofluorescent applications for the detection and treatment of tumorsand other abnormalities. These methods use light of wavelengths in theregion of 300–1300 nm. For example, optical coherence tomography (OCT),also referred to as “optical biopsy,” is an optical imaging techniquethat allows high resolution cross sectional imaging of tissuemicrostructure. OCT methods use wavelengths of about 1280 nm.

In various aspects of the invention, the dye-biomolecule conjugates areuseful for localized therapy for the detection of the presence orabsence of tumors and other pathologic tissues by monitoring the bloodclearance profile of the conjugates, for laser assisted guided surgery(LAGS) for the detection and treatment of small micrometastases oftumors, e.g., somatostatin subtype 2 (SST-2) positive tumors, uponlaparoscopy, and for diagnosis of atherosclerotic plaques and bloodclots.

In another embodiment, a therapeutic procedure comprises attaching aporphyrin or photodynamic therapy agent to a bioconjugate, and thenadministering light of an appropriate wavelength for detecting andtreating an abnormality.

The compositions of the invention can be formulated for enteral orparenteral administration. These formulations contain an effectiveamount of the dye-biomolecule conjugate along with conventionalpharmaceutical carriers and excipients appropriate for the type ofadministration contemplated. For example, parenteral formulationsadvantageously contain a sterile aqueous solution or suspension of theinventive conjugate, and may be injected directly, or may be mixed witha large volume parenteral composition or excipient for systemicadministration as is known to one skilled in the art. These formulationsmay also contain pharmaceutically acceptable buffers and/or electrolytessuch as sodium chloride.

Formulations for enteral administration may vary widely, as is wellknown in the art. In general, such formulations are aqueous solutions,suspensions or emulsions which contain an effective amount of adye-biomolecule conjugate. Such enteral compositions may includebuffers, surfactants, thixotropic agents, and the like. Compositions fororal administration may also contain flavoring agents and otheringredients for enhancing their organoleptic qualities.

The inventive compositions of the carbocyanine dye bioconjugates fordiagnostic uses are administered in doses effective to achieve thedesired effect. Such doses may vary widely, depending upon theparticular conjugate employed, the organs or tissues which are thesubject of the imaging procedure, the imaging equipment being used, andthe like. The compositions may be administered either systemically, orlocally to the organ or tissue to be imaged, and the patient is thensubjected to diagnostic imaging and/or therapeutic procedures.

The present invention is further detailed in the following Examples,which are offered by way of illustration and are not intended to limitthe scope of the invention in any manner.

EXAMPLE 1 Synthesis of Indocyaninebispropanoic Acid Dye (FIG. 1A, n=1)

A mixture of 1,1,2-trimethyl-[1H]-benz[e]indole (9.1 g, 43.58 mmoles)and 3-bromopropanoic acid (10.0 g, 65.37 mmoles) in 1,2-dichlorobenzene(40 ml) was heated at 110° C. for 12 hours. The solution was cooled toambient temperature. The red residue obtained was filtered and washedwith acetonitrile:diethyl ether (1:1^(v/v)) mixture. The solid obtainedwas dried at ambient temperature under vacuum to give 10 g (64%) oflight brown powder.

A portion of this solid (6.0 g; 16.56 mmoles), glutaconic aldehydedianilide hydrochloride (Lancaster Synthesis, Windham, N.H.) (2.36 g,8.28 mmoles), and sodium acetate trihydrate (2.93 g, 21.53 mmoles) inethanol (150 ml) were refluxed for 90 minutes. After evaporating thesolvent, 40 ml of a 2 N aqueous HCl was added to the residue. Themixture was centrifuged and the supernatant was decanted. This procedurewas repeated until the supernatant became nearly colorless. About 5 mlof a water:acetonitrile (3:2^(v/v)) mixture was added to the solidresidue and lyophilized to obtain 2 g of dark green flakes. The purityof the compound was established with ¹H-nuclear magnetic resonance(¹H-NMR) and liquid chromatography/mass spectrometry (LC/MS) as is knownto one skilled in the art.

EXAMPLE 2 Synthesis of Indocyaninebishexanoic Acid Dye (FIG. 1A, n=4)

A mixture of 1,1,2-trimethyl-[1H]-benz[e]indole (20 g, 95.6 mmoles) and6-bromohexanoic acid (28.1 g, 144.1 mmoles) in 1,2-dichlorobenzene (250ml) was heated at 110 C for 12 hours. The green solution was cooled toambient temperature and the brown solid precipitate that formed wascollected by filtration. After washing the solid with1,2-dichlorobenzene and diethyl ether, the brown powder obtained (24 g,64%) was dried under vacuum at ambient temperature. A portion of thissolid (4.0 g; 9.8 mmoles) glutacoaldehyde dianil monohydrochloride (1.4g, 5 mmoles) and sodium acetate trihydrate (1.8 g, 12.9 mmoles) inethanol (80 ml) were refluxed for 1 hour. After evaporating the solvent,20 ml of 2 N aqueous HCl was added to the residue. The mixture wascentrifuged and the supernatant was decanted. This procedure wasrepeated until the supernatant became nearly colorless. About 5 ml of awater:acetonitrile (3:2^(v/v)) mixture was added to the solid residueand lyophilized to obtain about 2 g of dark green flakes. The purity ofthe compound was established with ¹H-NMR and LC/MS.

EXAMPLE 3 Synthesis of Peptides

Peptides of this invention were prepared by similar procedures withslight modifications in some cases.

Octreotate, an octapeptide, has the amino acid sequenceD-Phe-Cys′-Tyr-D-Trp-Lys-Thr-Cys′-Thr (SEQ ID NO:1), wherein Cys′indicates the presence of an intramolecular disulfide bond between twocysteine amino acids. Octreotate was prepared by an automatedfluorenylmethoxycarbonyl (Fmoc) solid phase peptide synthesis using acommercial peptide synthesizer from Applied Biosystems (Model 432ASYNERGY Peptide Synthesizer). The first peptide cartridge contained Wangresin pre-loaded with Fmoc-Thr on a 25-μmole scale. Subsequentcartridges contained Fmoc-protected amino acids with side chainprotecting groups for the following amino acids: Cys(Acm), Thr(t-Bu),Lys(Boc), Trp(Boc) and Tyr(t-Bu). The amino acid cartridges were placedon the peptide synthesizer and the product was synthesized from the C-to the N-terminal position according to standard procedures. Thecoupling reaction was carried out with 75 μmoles of the protected aminoacids in the presence of2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU)/N-hydroxybenzotriazole (HOBt). The Fmoc protecting groups wereremoved with 20% piperidine in dimethylformamide.

After the synthesis was complete, the thiol group was cyclized withthallium trifluoroacetate and the product was cleaved from the solidsupport with a cleavage mixture containing trifluoroacetic acidwater:phenol:thioanisole (85:5:5:5^(v/v)) for 6 hours. The peptide wasprecipitated with t-butyl methyl ether and lyophilized withwater:acetonitrile (2:3^(v/v)). The peptide was purified by HPLC andanalyzed by LC/MS.

Octreotide, (D-Phe-Cys′-Tyr-D-Trp-Lys-Thr-Cys′-Thr-OH (SEQ ID NO:2)),wherein Cys′ indicates the presence of an intramolecular disulfide bondbetween two cysteine amino acids) was prepared by the same procedure asthat for octreotate with no modifications.

Bombesin analogs were prepared by the same procedure but cyclizationwith thallium trifluoroacetate was omitted. Side-chain deprotection andcleavage from the resin was carried out with 50 μl each ofethanedithiol, thioanisole and water, and 850 μl of trifluoroaceticacid. Two analogues were prepared:

Gly-Ser-Gly-Gln-Trp-Ala-Val-Gly-His- (SEQ ID NO: 3) Leu-Met-NH₂ andGly-Asp-Gly-Gln-Trp-Ala-Val-Gly-His- (SEQ ID NO: 4) Leu-Met-NH₂.

Cholecystokinin octapeptide analogs were prepared as described forOctreotate without the cyclization step. Three analogs were prepared:Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH₂ (SEQ ID NO:5);Asp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH₂ (SEQ ID NO:6); andD-Asp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH₂ (SEQ ID NO:7) wherein Nle isnorleucine.

Neurotensin analog (D-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu (SEQ ID NO:8)) wasprepared as described for Octreotate without the cyclization step.

EXAMPLE 4 Synthesis of Peptide-Dye Conjugates (FIG. 1B, n=1,R₁=Octreotate, R₂═R₁ or OH)

The method described below is for the synthesis of Octreotate-cyaninedye conjugates. Similar procedures were used for the synthesis of otherpeptide-dye conjugates.

Octreotate was prepared as described in Example 3, but the peptide wasnot cleaved from the solid support and the N-terminal Fmoc group of Phewas retained. The thiol group was cyclized with thalliumtrifluoroacetate and Phe was deprotected to liberate the free amine.

Bisethylcarboxymethylindocyanine dye (53 mg, 75 μmoles) was added to anactivation reagent consisting of a mixture 0.2 M HBTU/HOBt in DMSO (375μl), and 0.2 M diisopropylethylamine in DMSO (375 μl). The activationwas complete in about 30 minutes. The resin-bound peptide (25 μmoles)was then added to the dye. The coupling reaction was carried out atambient temperature for 3 hours. The mixture was filtered and the solidresidue was washed with DMF, acetonitrile and THF. After drying thegreen residue, the peptide was cleaved from the resin, and the sidechain protecting groups were removed with a mixture of trifluoroaceticacid: water:thioanisole:phenol (85:5:5:5^(v/v)). The resin was filteredand cold t-butyl methyl ether (MTBE) was used to precipitate thedye-peptide conjugate. The conjugate was dissolved in acetonitrile:water(2:3^(v/v)) and lyophilized.

The product was purified by HPLC to give themonooctreotate-bisethylcarboxymethylindocyanine dye (Cytate 1, 80%, n=1,R₂═OH) and the bisoctreotate-bisethylcarboxymethylindocyanine dye(Cytate 2, 20%, n=1, R₁═R₂).

The monooctreotate conjugate may be obtained almost exclusively (>95%)over the bis conjugate by reducing the reaction time to 2 hours. This,however, leads to an incomplete reaction, and the free octreotate mustbe carefully separated from the dye conjugate in order to avoidsaturation of the receptors by the non-dye conjugated peptide.

EXAMPLE 5 Synthesis of Peptide-Dye Conjugates (FIG. 1B, n=4R₁=octreotate, R₂═R₁ or OH)

Octreotate-bispentylcarboxymethylindocyanine dye was prepared asdescribed in Example 4 with some modifications.Bispentylcarboxymethylindocyanine dye (60 mg, 75 μmoles) was added to400 μl activation reagent consisting of 0.2 M HBTU/HOBt and 0.2 Mdiisopropylethylamine in DMSO. The activation was complete in about 30minutes and the resin-bound peptide (25 μmoles) was added to the dye.The reaction was carried out at ambient temperature for 3 hours. Themixture was filtered and the solid residue was washed with DMF,acetonitrile and THF. After drying the green residue, the peptide wascleaved from the resin and the side chain protecting groups were removedwith a mixture of trifluoroacetic acid:water:thioanisole:phenol(85:5:5:5^(v/v)). The resin was filtered and cold t-butyl methyl ether(MTBE) was used to precipitate the dye-peptide conjugate. The conjugatewas dissolved in acetonitrile:water (2:3^(v/v)) and lyophilized. Theproduct was purified by HPLC to giveoctreotate-1,1,2-trimethyl-[1H]-benz[e]indole propanoic acid conjugate(10%), monooctreotate-bispentylcarboxymethylindocyanine dye (Cytate 3,60%, n=4, R₂═OH) and bisoctreotate-bispentylcarboxymethylindocyanine dye(Cytate 4, 30%, n=4, R₁═R₂).

EXAMPLE 6 Synthesis of Peptide-Dye-Phototherapy Conjugates (FIG. 1B,n=4, R₁=Octreotate, R₂=HPPH) by Solid Phase

Bispentylcarboxymethylindocyanine dye (cyhex, 60 mg, 75 μmoles) indichloromethane is reacted with cyanuric acid fluoride (21 mg, 150mmoles) in the presence of pyridine (12 mg, 150 mmoles) for 30 minutesto produce an acid anhydride. One molar equivalent of2-[1-hexyloxyethyl]-2-devinylpyropheophorbide-a (HPPH, FIG. 1D,T=—NHC₂H₄NH₂) is added to the anhydride to form the cyhex-HPPH conjugatewith a free carboxylic acid group. This intermediate is added to anactivation reagent consisting of a 0.2 M solution of HBTU/HOBt in DMSO(400 μl), and a 0.2 M solution of diisopropylethylamine in DMSO (400μl). Activation of the carboxylic acid is complete in about 30 minutes.Resin-bound peptide (octreotate, 25 μmoles), prepared as described inExample 4, is added to the mixture. The reaction is carried out atambient temperature for 8 hours. The mixture is filtered and the solidresidue is washed with DMF, acetonitrile and THF. After drying the darkresidue at ambient temperature, the peptide derivative is cleaved fromthe resin and the side chain protecting groups are removed with amixture of trifluoroacetic acid:water:thioanisole:phenol(85:5:5:5^(v/v)). After filtering the resin, cold t-butyl methyl ether(MTBE) is used to precipitate the dye-peptide conjugate, which is thenlyophilized in acetonitrile:water (2:3^(v/v)).

EXAMPLE 7 Synthesis of Peptide-Dye-Phototherapy Conjugates (FIG. 1B,n=4, R₁=Octreotide R₂=HPPH) by Solution Phase

Derivatized HPPH ethylenediamine (FIG. 1D, T=—NHC₂H₄NH₂; 1.1 molarequivalents) and lysine(trityl)⁴ octreotide (1.2 molar equivalents) wereadded to a solution of bis(pentafluorophenyl) ester of cyhex (1 molarequivalent) in DMF. After stirring the mixture for 8 hours at ambienttemperature, cold t-butyl methyl ether was added to precipitate thepeptide conjugate. The crude product was purified by high performanceliquid chromatography (HPLC).

EXAMPLE 8 Synthesis of Peptide-Dye-Phototherapy Conjugates (FIG. 1C,n=4, R₁=K⁰-Octreotate, R₂=HPPH, R₃=OH) by Solid Phase

Orthogonally protected Fmoc-lysine(Mtt)⁰ Octreotate was prepared on asolid support, as described in Examples 3 and 4. The Fmoc group ofFmoc-lysine(Mtt)⁰ is removed from the solid support with 20% piperidinein DMF. HPPH (FIG. 1D, T=—OH), pre-activated with HBTU coupled to thefree α-amino group of lysine.

EXAMPLE 9 Imaging of Tumor Cell Lines with Indocyanine Green

A non-invasive in vivo fluorescence imaging apparatus was employed toassess the efficacy of indocyanine green (ICG) in three different rattumor cell lines of the inventive contrast agents developed for tumordetection in animal models. A LaserMax Inc. laser diode of nominalwavelength 780 nm and nominal power of 40 mW was used. The detector wasa Princeton Instruments model RTE/CCD-1317-K/2 CCD camera with aRodenstock 10 mm F2 lens (stock #542.032.002.20) attached. An 830 nminterference lens (CVI Laser Corp., part #F10-830-4-2) was mounted infront of the CCD input lens, such that only emitted fluorescent lightfrom the contrast agent was imaged.

Three tumor cell lines, DSL 6/A (pancreatic), Dunning R3327-H(prostate), and CA20948 (pancreatic), which are rich in somatostatin(SST-2) receptors were induced into male Lewis rats by solid implanttechnique in the left flank area (Achilefu et al., Invest. Radiology,2000, pp. 479–485). Palpable masses were detected nine days postimplant.

The animals were anesthetized with xylazine:ketamine:acepromazine(1.5:1.5:0.5^(v/v)) at 0.8 ml/kg via intramuscular injection. The leftflank was shaved to expose the tumor and surrounding surface area. A21-gauge butterfly needle equipped with a stopcock connected to twosyringes containing heparinized saline was placed into the tail vein ofthe rat. Patency of the vein was checked prior to administration of ICG.Each animal was administered a 0.5 ml dose of a 0.42 mg/ml solution ofICG in saline.

Two of the cell lines, DSL 6/A (pancreatic) and Dunning R3327-H(prostate) which are rich in somatostatin (SST-2) receptors indicatedslow perfusion of the agent into the tumor over time. Images were takenat 2 minutes and 30 minutes post administration of ICG. Reasonableimages were obtained for each. The third line, CA20948 (pancreatic),indicated only a slight and transient perfusion that was cleared afteronly 30 minutes post injection. This indicated that there was nonon-specific localization of ICG into this tumor line compared to theother two lines which suggested a vastly different vascular architecturefor this type of tumor (FIG. 2). The first two tumor lines (DSL 6/A andR3327-H) were not as highly vascularized as CA20948 which is also richin somatostatin (SST-2) receptors. Consequently, the detection andretention of a dye in the CA20948 tumor model is an important index ofreceptor-mediated specificity.

EXAMPLE 10 Imaging of Rat Pancreatic Acinar Carcinoma (CA20948) withCytate 1

The peptide, octreotate, is known to target somatostatin (SST-2)receptors. Therefore, the cyano-octreotates conjugate, Cytate 1, wasprepared as described in Example 4. The pancreatic acinar carcinoma,CA20948, was induced into male Lewis rats as described in Example 9.

The animals were anesthetized with xylazine:ketamine:acepromazine(1.5:1.5:0.5^(v/v)) at 0.8 ml/kg via intramuscular injection. The leftflank was shaved to expose the tumor and surrounding surface area. A21-gauge butterfly needle equipped with a stopcock connected to twosyringes containing heparinized saline was placed into the tail vein ofthe rat. Patency of the vein was checked prior to administration ofCytate 1 via the butterfly apparatus. Each animal was administered a 0.5ml dose of a 1.0 mg/ml solution of Cytate 1 in 25%^((v/v))dimethylsulfoxide/water.

Using the CCD camera apparatus, dye localization in the tumor wasobserved. Usually, an image of the animal was taken pre-injection ofcontrast agent, and the pre-injection image was subsequently subtracted(pixel by pixel) from the post-injection images to remove background.However, the background subtraction was not done if the animal had beenremoved from the sample area and was later returned for imaging severalhours post injection. These images demonstrated the specificity ofcytate 1 for the SST-2 receptors present in the CA20948 rat tumor model.

At one minute post administration of cytate 1 the fluorescent imagesuggested the presence of the tumor in the left flank of the animal(FIG. 3 a). At 45 minutes post administration, the image showed greenand yellow areas in the left and right flanks and in the tail, however,there was a dark blue/blue green area in the left flank (FIG. 3 b). AT27 hours post administration of the conjugate, only the left flankshowed a blue/blue green fluorescent area (FIG. 4).

Individual organs were removed from the CA20948 rat which was injectedwith cytate 1 and were imaged. High uptake of the conjugate was observedin the pancreas, adrenal glands and tumor tissue. Significant loweruptake was observed in heart, muscle, spleen and liver (FIG. 5). Theseresults correlated with results obtained using radiolabeled octreotatein the same rat model system (M. de Jong, et al. Cancer Res. 1998, 58,437–441).

EXAMPLE 11 Imaging of Rat Pancreatic Acinar Carcinoma (AR42-J) withBombesinate

The AR42-J cell line is derived from exocrine rat pancreatic acinarcarcinoma. It can be grown in continuous culture or maintained in vivoin athymic nude mice, SCID mice, or in Lewis rats. This cell line isparticularly attractive for in vitro receptor assays, as it is known toexpress a variety of hormone receptors including cholecystokinin (CCK),epidermal growth factor (EGF), pituitary adenylate cyclase activatingpeptide (PACAP), somatostatin (sst₂) and bombesin.

In this model, male Lewis rats were implanted with solid tumor materialof the AR42-J cell line in a manner similar to that described in Example9. Palpable masses were present 7 days post implant, and imaging studieswere conducted on animals when the mass had achieved approximately 2 to2.5 g (10–12 days post implant).

FIG. 6 shows the image obtained with this tumor model at 22 hours postinjection of bombesinate. Uptake of bombesinate was similar to thatdescribed in Example 10 for uptake of cytate 1 with specificlocalization of the bioconjugate in the tumor.

EXAMPLE 12 Imaging of Rat Pancreatic Acinar Carcinoma (CA20948) withCytate 1 by Fluorescence Endoscopy

Fluorescence endoscopy is suitable for tumors or other pathologicconditions of any cavity of the body. It is very sensitive and is usedto detect small cancerous tissues, especially in the lungs andgastrointestinal (GI) system. Methods and procedures for fluorescenceendoscopy are well-documented [Tajiri H., et al. Fluorescent diagnosisof experimental gastric cancer using a tumor-localizing photosensitizer.Cancer Letters (1997) 111, 215–220; Sackmann M., Fluorescence diagnosisin GI endoscop, Endoscopy (2000) 32, 977–985, and references therein].

The fluorescence endoscope consists of a small optical fiber probeinserted through the working channel of a conventional endoscope. Somefibers within this probe deliver the excitation light at 780 nm andothers detect the fluorescence from the injected optical probe at 830nm. The fluorescence intensity is displayed on a monitor.

Briefly, the CA20948 rat pancreatic tumor cells which areover-expressing somatostatin receptor are injected into the submucosa ofa Lewis rat. The tumor is allowed to grow for two weeks. The rat is thenanesthetized with xylazine:ketamine:acepromazine (1.5:1.5:0.5/^(v/v)) at0.8 mL/kg via intramuscular injection. Cytate is injected in the tailvein of the rat and 60 minutes post-injection, the endoscope is insertedinto the GI tract. Since cytate localizes in CA20948, the fluorescenceintensity in the tumor is much higher than in the surrounding normaltissues. Thus, the relative position of the tumor is determined byobserving the image on a computer screen.

EXAMPLE 13 Imaging of Rat Pancreatic Acinar Carcinoma (CA20948) withCytate 1 by Photoacoustic Technique

The photoacoustic imaging technique combines optical and acousticimaging to allow better diagnosis of pathologic tissues. The preferredacoustic imaging method is ultrasonography where images are obtained byirradiating the animal with sound waves. The dual ultrasonography andoptical tomography enables the imaging and localization of pathologicconditions (e.g., tumors) in deep tissues. To enhance the imaging,cytate is incorporated into ultrasound contrast material. Methods forthe encapsulation of gases in biocompatible shells that are used as thecontrast material are described in the literature [Mizushige K., et al.,Enhancement of ultrasound-accelerated thrombolysis by echo contrastagents: dependence on microbubble structure, Ultrasound in Med. & Biol.(1999), 25, 1431–1437]. Briefly, perfluorocarbon gas (e.g.,perfluorobutane) is bubbled into a mixture of normal saline:propyleneglycol:glycerol (7:1.5:1.5v/v/v) containing 7 mg/ml ofcytate:dipalmitoylphosphatidylcholine:dipalmitoylphosphatidic acid, anddipalmitoylphosphatidylethanolamine-PEG 5,000 (1:7:1:1 mole %). TheCA20948 tumor bearing Lewis rat is injected with 1 ml of themicrobubbles and the agent is allowed to accumulate in the tumor. Anoptical image is obtained by exciting the near infrared dye at 780 nmand detecting the emitted light at 830 nm, as described in Examples9–11. Ultrasonography is performed by irradiating the rat with soundwaves in the localized tumor region and detecting the reflected sound asdescribed in the literature [Peter J. A. Frinking, Ayache Bouakaz, JohanKirkhorn, Folkert J. Ten Cate and Nico de Jong, Ultrasound contrastimaging: current and new potential methods, Ultrasound in Medicine &Biology (2000) 26, 965–975].

EXAMPLE 14 Photodynamic Therapy (PDT) and Localized Therapy of RatPancreatic Acinar Carcinoma (CA20948) with Cytate-PDT AgentBioconjugates

The method for photodynamic therapy is well documented in the literature[Rezzoug H., et al. In Vivo Photodynamic Therapy with meso-Tetra(m-hydroxyphenyl)chlorin (mTHPC): Influence of Light Intensity andOptimization of Photodynamic Efficiency, Proc. SPIE (1996), 2924,181–186; Stranadko E., et al. Photodynamic Therapy of Recurrent Cancerof Oral Cavity, an Alternative to Conventional Treatment, Proc. SPIE(1996), 2924, 292–297]. A solution of the peptide-dye-phototherapybioconjugate is prepared as described in Example 7 (5 μmol/mL of 15%DMSO in water, 0.5 mL) and is injected into the tail vein of thetumor-bearing rat. The rat is imaged 24 hours post injection asdescribed in Examples 9–11 to localize the tumor. Once the tumor regionis localized, the tumor is irradiated with light of 700 nm (whichcorresponds to the maximum absorption wavelength of HPPH, the componentof the conjugate that effects PDT). The energy of radiation is 10 J/cm²at 160 mW/cm². The laser light is transmitted through a fiber optic,which is directed to the tumor. The rat is observed for 7 days and anydecrease in tumor volume is noted. If the tumor is still present, asecond dose of irradiation is repeated as described above until thetumor is no longer palpable.

For localized therapy, a diagnostic amount of cytate (0.5 mL/0.2 kg rat)is injected into the tail vein of the tumor-bearing rat and opticalimages are obtained as described in Examples 9–11. A solution of thepeptide-dye-phototherapy bioconjugate is prepared as described inExample 7 (5 μmol/mL of 15% DMSO in water, 1.5 mL) and is injecteddirectly into the tumor. The tumor is irradiated as described above.

EXAMPLE 15 Photodiagnosis with Atherosclerotic Plaques and Blood Clots

A solution of a peptide-dye-bioconjugate for targeting atheroscleroticplaques and associated blood clots is prepared as described in Example7. The procedure for injecting the bioconjugate and subsequentlocalization and diagnosis of the plaques and clots is performed asdescribed in Example 14.

While the invention has been disclosed by reference to the details ofpreferred embodiments of the invention, it is to be understood that thedisclosure is intended in an illustrative rather than in a limitingsense, as it is contemplated that modifications will readily occur tothose skilled in the art, within the spirit of the invention and thescope of the appended claims.

1. A compound having the carbocyanine dye bioconjugate of formula 2

wherein W₁ and W₂ are S; Y₁, Y₂, Z₁, and Z₂ are independently selectedfrom the group consisting of hydrogen, tumor-specific agent,phototherapy agent, —CONH-Bm, —NHCO-Bm, —(CH₂)_(a)—CONH-Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH-Bm, —(CH₂)_(a)—NHCO-Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO-Bm, —(CH₂)_(a)—N(R¹²)—(CH₂)_(b)—CONH-Bm,(CH₂)_(a)—N(R¹²)—(CH₂)_(c)—NHCO-Bm,—(CH₂)_(a)—N(R¹²)—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH-Bm,—(CH₂)_(a)—N(R¹²)—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO-Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R¹²)—(CH₂)_(a)—CONH-Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R¹²)—(CH₂)_(a)—NHCO-Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R¹²)—CH₂—(CH₂OCH₂)_(d)—CONH-Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R¹²)—CH₂—(CH₂OCH₂)_(d)—NHCO-Bm, —CONH-Dm,—NHCO-Dm, —(CH₂)_(a)—CONH-Dm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH-Dm,—(CH₂)_(a)—NHCO-Dm, —CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO-Dm,—(CH₂)_(a)—N(R¹²)—(CH₂)_(b)—CONH-Dm,—(CH₂)_(a)—N(R¹²)—(CH₂)_(c)—NHCO-Dm,—(CH₂)_(a)—N(R¹²)—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH-Dm,—(CH₂)_(a)—N(R¹²)—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO-Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R¹²)—(CH₂)_(a)—CONH-Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R¹²)—(CH₂)_(a)—NHCO-Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R¹²)—CH₂—(CH₂OCH₂)_(d)—CONH-Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R¹²)—CH₂—(CH₂OCH₂)_(d)—NHCO-Dm, —(CH₂)_(a)—NR¹²R¹³, and —CH₂(CH₂OCH₂)_(b)—CH₂N R¹²R¹³; K₁ and K₂ are independentlyselected from the group consisting of C₅–C₃₀ aryl, C₁–C₃₀ alkoxyl,C₁–C₃₀ polyalkoxyalkyl, C₁–C₃₀ polyhydroxyalkyl, C₅–C₃₀ polyhydroxyaryl,C₁–C₃₀ aminoalkyl, saccharide, peptide, —CH₂(CH₂OCH₂)_(b)—CH₂—,—(CH₂)_(a)—CO—, —(CH₂)_(a)—CONH, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—,—(CH₂)_(a)—NHCO—, —CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—, —(CH₂)_(a)—O—, and—CH₂—(CH₂OCH₂)_(b)—CO—; X₁ and X₂ are single bonds, or are independentlyselected from the group consisting of nitrogen, saccharide, —CR¹⁴—,—CR¹⁴R¹⁵, —NR¹⁶R¹⁷; C₅–C₃₀ aryl; Q is a single bond or is selected fromthe group consisting of —O—, —S—, —Se—, and —NR¹⁶; a₁ and b₁independently vary from 0 to 5; R¹², R¹³, and R¹⁸ to R³¹ areindependently selected from the group consisting of hydrogen, C₁–C₁₀alkyl, C₅–C₂₀ aryl, C₁–C₁₀ alkoxyl, C₁–C₁₀ polyalkoxyalkyl, C₁–C₂₀polyhydroxyalkyl, C₅–C₂₀ polyhydroxyaryl, C₁–C₁₀ aminoalkyl, cyano,nitro, halogen, saccharide, peptide, —CH₂(CH₂OCH₂)_(b)—CH₂—OH,—(CH₂)_(a)—CO₂H, —(CH₂)_(a)—CONH-Bm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH-Bm,—(CH₂)_(a)—NHCO-Bm, —CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO-Bm, —(CH₂)_(a)—OH and—CH₂—(CH₂OCH₂)_(b)—CO₂H; R¹⁴ to R¹⁷ are independently selected from thegroup consisting of hydrogen, C₁–C₁₀ alkyl, C₅–C₂₀ aryl, C₁–C₁₀ alkoxyl,C₁–C₁₀ polyalkoxyalkyl, C₁–C₂₀ polyhydroxyalkyl, C₅–C₂₀ polyhydroxyaryl,C₁–C₁₀ aminoalkyl, saccharide, peptide, —CH₂(CH₂OCH₂)_(b)—CH₂—,—(CH₂)_(a)—CO—, —(CH₂)_(a)—CONH—, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—,—(CH₂)_(a)—NHCO—, —CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—, —(CH₂)_(a)—O—, and—CH₂—(CH₂OCH₂)_(b)—CO—; Bm and Dm are independently selected from thegroup consisting of bioactive peptide, protein, cell, antibody, antibodyfragment, saccharide, glycopeptide, peptidomimetic, drug, drug mimic,hormone, metal chelating agent, radioactive or nonradioactive metalcomplex, echogenic agent, photoactive molecule, and phototherapy agent;a and c independently vary from 1 to 20; b and d independently vary from1 to
 100. 2. The compound of claim 1 wherein W₁ and W₂ are S; Y₁ and Y₂are selected from the group consisting of hydrogen, tumor-specificagent, —CONH-Bm, —NHCO-Bm, —(CH₂)_(a)—CONH-Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH-Bm, —(CH₂)_(a)—NHCO-Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO-Bm, —(CH₂)_(a)—NR¹²R¹³, and—CH₂(CH₂OCH₂)_(b)—CH₂NR¹²R¹³; Z₁ and Z₂ are independently selected fromthe group consisting of hydrogen, phototherapy agent, —CONH-Dm,—NHCO-Dm, —(CH₂)_(a)—CONH-Dm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH-Dm,—(CH₂)_(a)—NHCO-Dm, —CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO-Dm, —(CH₂)_(a)—N R¹²R¹³,and —CH₂(CH₂OCH₂)_(b)—CH₂N R¹²R¹³; K₁ and K₂ are independently selectedfrom the group consisting of C₅–C₂₀ aryl, C₁–C₂₀ alkoxyl, C₁–C₂₀aminoalkyl, —(CH₂)_(a)—CO—, —(CH₂)_(a)—CONH,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—, —(CH₂)_(a)—NHCO—,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—, and —CH₂—(CH₂OCH₂)_(b)—CO—; X₁ and X₂ aresingle bonds, or are independently selected from the group consisting ofnitrogen, —CR¹⁴—, —CR¹⁴R¹⁵, and —NR¹⁶R¹⁷; Q is a single bond or isselected from the group consisting of —O—, —S—, and —NR¹⁸; a₁ and b₁independently vary from 0 to 3; Bm is selected from the group consistingof bioactive peptide containing 2 to 30 amino acid units, protein,antibody fragment, mono- and oligosaccharide; Dm is selected from thegroup consisting of photosensitizer, photoactive molecule, andphototherapy agent; a and c independently vary from 1 to 10; b and dindependently vary from 1 to
 30. 3. The compound of claim 2 wherein eachW₁ and W₂ is S; each K₁ and K₂ is —(CH₂)₄CO—; each Q, X₁ and X₂ is asingle bond; each R¹⁹ to R³¹, Y₁ and Z₁ is H; Y₂ is a tumor-specificagent; and Z₂ is a phototherapy agent.
 4. The compound according toclaim 3 wherein the said tumor-specific agent is a bioactive peptidecontaining 2 to 30 amino acid units.
 5. The compound according to claim4 wherein the said tumor-specific agent is octreotate and bombesin(7–14).
 6. The compound according to claim 3 wherein the saidphototherapy agent is a photosensitizer.
 7. The compound according toclaim 6 wherein the said photosensitizer is2-[1-hexyloxyethyl]-2-devinylpyropheophorbide-a.
 8. A method forperforming a diagnostic and therapeutic procedure comprisingadministering to an individual an effective amount of the composition ofcyanine dye bioconjugate of Formula 2

wherein W₁ and W₂ are S; Y₁, Y₂, Z₁, and Z₂ are independently selectedfrom the group consisting of hydrogen, tumor-specific agent,phototherapy agent, —CONH-Bm, —NHCO-Bm, —(CH₂)_(a)—CONH-Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH-Bm, —(CH₂)_(a)—NHCO-Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO-Bm, —(CH₂)_(a)—N(R¹²)—(CH₂)_(b)—CONH-Bm,—(CH₂)_(a)—N(R¹²)—(CH₂)_(c)—NHCO-Bm,—(CH₂)_(a)—N(R¹²)—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH-Bm,—(CH₂)_(a)—N(R¹²)—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO-Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R¹²)—(CH₂)_(a)—CONH-Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R¹²)—(CH₂)_(a)—NHCO-Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R¹²)—CH₂—(CH₂OCH₂)_(d)—CONH-Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R¹²)—CH₂—(CH₂OCH₂)_(d)—NHCO-Bm, —CONH-Dm,—NHCO-Dm, —(CH₂)_(a)—CONH-Dm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH-Dm,—(CH₂)_(a)—NHCO-Dm, —CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO-Dm,—(CH₂)_(a)—N(R¹²)—(CH₂)_(b)—CONH-Dm,—(CH₂)_(a)—N(R¹²)—(CH₂)_(c)—NHCO-Dm,—(CH₂)_(a)—N(R¹²)—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH-Dm,—(CH₂)_(a)—N(R¹²)—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO-Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R¹²)—(CH₂)_(a)—CONH-Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R¹²)—(CH₂)_(a)—NHCO-Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R¹²)—CH₂—(CH₂OCH₂)_(d)—CONH-Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R¹²)—CH₂—(CH₂OCH₂)_(d)—NHCO-Dm, —(CH₂)_(a)—NR¹²R¹³, and —CH₂(CH₂OCH₂)_(b)—CH₂N R¹²R¹³; K₁ and K₂ are independentlyselected from the group consisting of C₁–C₃₀ alkyl, C₅–C₃₀ aryl, C₁–C₃₀alkoxyl, C₁–C₃₀ polyalkoxyalkyl, C₁–C₃₀ polyhydroxyalkyl, C₅–C₃₀polyhydroxyaryl, C₁–C₃₀ aminoalkyl, saccharide, peptide,—CH₂(CH₂OCH₂)_(b)—CH₂—, —(CH₂)_(a)—CO—, —(CH₂)_(a)—CONH,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—, —(CH₂)_(a)—NHCO—,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—, —(CH₂)_(a)—O—, and —CH₂—(CH₂OCH₂)_(b)—CO—;X₁ and X₂ are single bonds, or are independently selected from the groupconsisting of nitrogen, saccharide, —CR¹⁴—, —CR¹⁴R¹⁵, —NR¹⁶R¹⁷; C₅–C₃₀aryl; Q is a single bond or is selected from the group consisting of—O—, —S—, —Se—, and —NR¹⁸; a₁ and b₁ independently vary from 0 to 5;R¹², R¹³, and R¹⁸ to R³¹ are independently selected from the groupconsisting of hydrogen, C₁–C₁₀ alkyl, C₅–C₂₀ aryl, C₁–C₁₀ alkoxyl,C₁–C₁₀ polyalkoxyalkyl, C₁–C₂₀ polyhydroxyalkyl, C₅–C₂₀ polyhydroxyaryl,C₁–C₁₀ aminoalkyl, cyano, nitro, halogen, saccharide, peptide,—CH₂(CH₂OCH₂)_(b)—CH₂—OH, —(CH₂)_(a)—CO₂H, —(CH₂)_(a)—CONH-Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH-Bm, —(CH₂)_(a)—NHCO-Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO-Bm, —(CH₂)_(a)—OH and—CH₂—(CH₂OCH₂)_(b)—CO₂H; R¹⁴ to R¹⁷ are independently selected from thegroup consisting of hydrogen, C₁–C₁₀ alkyl, C₅–C₂₀ aryl, C₁–C₁₀ alkoxyl,C₁–C₁₀ polyalkoxyalkyl, C₁–C₂₀ polyhydroxyalkyl, C₅–C₂₀ polyhydroxyaryl,C₁–C₁₀ aminoalkyl, saccharide, peptide, —CH₂(CH₂OCH₂)_(b)—CH₂—,—(CH₂)_(a)—CO—, —(CH₂)_(a)—CONH, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—,—(CH₂)_(a)—NHCO—, —CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—, —(CH₂)_(a)—O—, and—CH₂—(CH₂OCH₂)_(b)—CO—; Bm and Dm are independently selected from thegroup consisting of bioactive peptide, protein, cell, antibody, antibodyfragment, saccharide, glycopeptide, peptidomimetic, drug, drug mimic,hormone, metal chelating agent, radioactive or nonradioactive metalcomplex, echogenic agent, photoactive molecule, and phototherapy agent;a and c independently vary from 1 to 20; b and d independently vary from1 to 100; and thereafter, performing said procedure.
 9. The method forperforming the diagnostic and therapeutic procedure of claim 8comprising administering to an individual an effective amount of thecomposition of cyanine dye bioconjugate wherein W₁ and W₂ are S; Y₁ andY₂ are selected from the group consisting of hydrogen, tumor-specificagent, —CONH-Bm, —NHCO-Bm, —(CH₂)_(a)—CONH-Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH-Bm, —(CH₂)_(a)—NHCO-Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO-Bm, —(CH₂)_(a)—NR¹²R¹³, and—CH₂(CH₂OCH₂)_(b)—CH₂NR¹²R¹³; Z₁ and Z₂ are independently selected fromthe group consisting of hydrogen, phototherapy agent, —CONH-Dm,—NHCO-Dm, —(CH₂)_(a)—CONH-Dm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH-Dm,—(CH₂)_(a)—NHCO-Dm, —CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO-Dm, —(CH₂)_(a)—N R¹²R¹³,and —CH₂(CH₂OCH₂)_(b)—CH₂N R¹²R¹³; K₁ and K₂ are independently selectedfrom the group consisting of C₁–C₁₀ alkyl, C₅–C₂₀ aryl, C₁–C₂₀ alkoxyl,C₁–C₂₀ aminoalkyl, —(CH₂)_(a)—CO—, —(CH₂)_(a)—CONH—,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—, —(CH₂)_(a)—NHCO—,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—, and —CH₂—(CH₂OCH₂)_(b)—CO—; X₁ and X₂ aresingle bonds, or are independently selected from the group consisting ofnitrogen, —CR¹⁴—, —CR¹⁴R¹⁵, and —NR¹⁶R¹⁷; Q is a single bond or isselected from the group consisting of —O—, —S—, and —NR¹⁸; a₁ and b₁independently vary from 0 to 3; Bm is selected from the group consistingof bioactive peptide containing 2 to 30 amino acid units, protein,antibody fragment, mono- and oligosaccharide; Dm is selected from thegroup consisting of photosensitizer, photoactive molecule, andphototherapy agent; a and c independently vary from 1 to 10; b and dindependently vary from 1 to
 30. 10. The method for performing thediagnostic and therapeutic procedure of claim 9 comprising administeringto an individual an effective amount of the composition of cyanine dyebioconjugate wherein each W₁ and W² are S; each K₁ and K₂ is —(CH₂)₄CO—;each Q, X₁ and X₂ is a single bond; each R¹⁹ to R³¹, Y₁ and Z₁ is H; Y₂is a tumor-specific agent; and Z₂ is a phototherapy agent.
 11. Themethod for performing the diagnostic and therapeutic procedure of claim10 comprising administering to an individual an effective amount of thecomposition of cyanine dye bioconjugate wherein the said tumor-specificagent is a bioactive peptide containing 2 to 30 amino acid units. 12.The method for performing the diagnostic and therapeutic procedure ofclaim 11 comprising administering to an individual an effective amountof the composition of cyanine dye bioconjugate wherein the saidtumor-specific agent is octreotate and bombesin (7–14).
 13. The methodfor performing the diagnostic and therapeutic procedure of claim 10comprising administering to an individual an effective amount of thecomposition of cyanine dye bioconjugate wherein the said phototherapyagent is a photosensitizer.
 14. The method for performing the diagnosticand therapeutic procedure of claim 13 comprising administering to anindividual an effective amount of the composition of cyanine dyebioconjugate wherein the said photosensitizer is2-[1-hexyloxyethyl]-2-devinylpyropheophorbide-a.
 15. The method of claim8 wherein said procedure utilizes light of wavelength in the region of300–1300 nm.
 16. The method of claim 8 wherein the diagnostic procedureis optical tomography.
 17. The method of claim 8 wherein said diagnosticprocedure is fluorescence endoscopy.
 18. The method of claim 8 whereinsaid procedure further comprises a step of imaging and therapy whereinsaid imaging and therapy is selected from the group consisting ofabsorption, light scattering, photoacoustic and sonofluoresencetechnique.
 19. The method of claim 8 wherein said procedure is fordiagnosing and treating atherosclerotic plaques and blood clots.
 20. Themethod of claim 8 wherein said procedure comprises administeringlocalized therapy.
 21. The method of claim 8 wherein said therapeuticprocedure comprises photodynamic therapy.
 22. The method of claim 8wherein said therapeutic procedure comprises laser assisted guidedsurgery (LAGS) for the detection and treatment of micrometastases.